MicroRNA-mediated gene regulation controls the levels of many proteins in eukaryotic cells. Gene transcripts are silenced after binding to a complementary ~21-25 nucleotide guide RNA that is associated with proteins including Argonaute (Ago), which together constitute the functional core of the RNA- induced silencing complex (RISC). Upon binding to an mRNA, the RISC interferes with its translation and/or triggers mRNA degradation, thus preventing protein synthesis. Each guide sequence, which specifies a gene suppressed by this mechanism, is produced and incorporated into the RISC by proteins of the RISC loading complex (RLC). The focus of the research herein is to investigate the RLC proteins Dicer and TRBP, both of which occur at reduced or undetectable levels in cancer cells according to recent findings. Furthermore, TRBP is known to be oncogenic. MicroRNA misregulation is known to cause cancer, but the mechanism is poorly understood. Elucidation of RLC components will provide clues to the connection between microRNA and cancer. This project will determine the three dimensional structures of Dicer and TRBP in the context of their interactions with each other and with RLC substrate dsRNAs. Because these proteins contribute to an essential means of gene regulation in eukaryotes, completion of the following aims will illuminate the mechanisms of microRNA-mediated gene regulation and enable manipulation of these pathways for therapeutic purposes. Furthermore, this project will provide mechanistic insight into the connections between microRNA production and cancer. The focus of the research presented herein is to investigate the miRNA- processing proteins Dicer and TRBP via structural study of each protein in the context of their interactions with each other and with substrate double-stranded RNA (dsRNA). The structures and interaction modes of TRBP and Dicer will illuminate the mechanism of miRNA action, clarifying connections to cancer and enabling manipulation of these pathways for therapeutic purposes. The aims of this project are: (1) to determine TRBP's structure and interactions with other components of the RLC;(2) to investigate Dicer's interactions with other components of the RLC. The TRBP and Dicer proteins will be studied using structural biology techniques such as NMR spectroscopy and X-ray crystallography. A novel approach will be taken in the study of TRBP: instead of targeting the entire protein, individual domains will be studied. Interactions with substrate dsRNA will be screened to identify the best conditions for further structural study of protein-RNA complexes. As with TRBP, truncated Dicer constructs will serve as structural targets in addition to the full-length protein. An additional goal will be study of Dicer bound to its miRNA substrate, whose interaction details remain unknown. PUBLIC HEALTH RELEVANCE: This research focuses on a recently discovered pathway for gene regulation based on genetic messages called microRNAs. Some of the cellular machinery responsible for microRNA processing is known to be involved in the formation of tumors, but the connection is currently unclear. We will study the structures of such microRNA-processing machinery to uncover their link to cancer and to help inform new gene therapy techniques that employ the cellular machinery in question.